The polymerase chain reaction is a technique that is used for increasing the quantity of a nucleic acid target. By thermal cycling a reaction cocktail of short primer sequences, free nucleotides and DNA polymerase, a template strand can be copied. For the polymerase chain reaction (PCR), temperature plateaus are often required: a high temperature for denaturation of the amplicon of interest, a low temperature for the binding of primers to the template, and an intermediate temperature for the synthesis of a complement strand.
Specificity of the binding between the primers and the template allows for selective amplification of intended targets. For instance, a typical quantitative polymerase chain reaction is run to “saturation,” wherein the amplification reaction is run to the point at which at least one of the reactants such as primers, polymerase, probe, or free nucleotides is exhausted. When fluorescent reporters such as TaqMan probes, FRET probes, or intercalating dyes are used to interpret the extent of the DNA amplification, a fluorescence curve could be generated. The sigmoidal signature of a “saturation” fluorescence curve often occurs when the reporter probe concentrations are depleted at an ever-increasing rate.
However, this reaction (and by extension, its signature fluorescence) is limited by all reactant concentrations, including the PCR primers. As an example, when a single set of primers is used in equal concentrations, and when one primer is more efficiently extended than the other, the reaction becomes rate limited. This results in a linear, as compared to an exponential, amplification of DNA.